Histoplasma capsulatum is a classic dimorphic fungal pathogen that initially infects mammalian hosts as a mold and then undergoes a complete transition to a yeast. The conversion, can be completely reproduced in vitro merely by raising the temperature from 25 degrees Celcius to 37 degrees Celcius, allowing us to mimic this natural switch between a form dedicated to a saprophytic lifestyle and a form dedicated to a parasitic lifestyle. Correspondingly, we anticipate that differences in gene expression between the two forms growing in vitro will not only relate to morphology, but also to phenotypes important for these alternative lifestyles. In this research plan, we will test the hypothesis that intracellular parasitism by H. capsulatum in rive is dependent on genes and gene products that are selectively upregulated during the conversion to or maintenance of the yeast phase in vitro. Our goal is to focus on genes that are transcriptionally more active during yeast growth (compared to mold growth) or that have a demonstrated importance for expression of a yeast phase-specific phenotype. Our specific aims are to: I. Define structure-activity relationships for the secreted calcium-binding protein (CBP). Produced in large amounts by yeasts growing in vitro or within macrophages, CBP is a proven virulence factor with an unproven me_anism. We plan to use molecular genetics in a rationally designed study to link structural features with specific functions related to virulence, dimorphism, and calcium binding. II. Characterize the regulation of known yeast phase-specific products. We will use a combination of forwardand reverse genetic strategies to understand the regulation of two yeast phase-specific products: CBP, which is unique to H. capsulatum, and e Alpha-(1,3)-glucan, a common feature of organisms causing systemic mycoses. III. Generate physically marked mutants with phenotypes in yeast phase-specific characteristics. Combining Agrobacterium-mediated mutagenesis with a series of simple in vitro screens, we wilt identify and characterize mutants affiScted in a variety of virulence-related, phase-specific phenotypes such as dimorphism, calcium dependence, cell wall composition, pH modulation, and redox modulation.